Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/72—Nitrogen atoms
  • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• This invention relates to a new class of compounds, which are potent and highly selective inhibitors of factor Xa, both isolated and when assembled in a complex with other proteins.
• the invention relates to a new class of compounds, their pharmaceutically acceptable salts, and pharmaceutically acceptable compositions thereof, which are useful as potent and selective inhibitors of blood coagulation in humans and other mammals.
• the invention relates to new methods for using new classes of agents for treating diseases associated with coagulation disorders in humans and other mammals.
• Hemostasis the control of bleeding, is effectuated either by surgical means or by affecting the physiological state of blood vessels or the coagulation.
• This invention particularly concerns blood coagulation and its role in maintaining the functioning of the human organism after injury, inflammation, disease, congenital defect, dysfunction, or other disruption.
• Thrombin is a key protein responsible for the coagulation.
• Thrombin plays the main role in thrombosis due to its ability to catalyze the conversion of fibrinogen into fibrin.
• Direct and indirect inhibitors of thrombin till recently have been the focus of a variety of anticoagulant strategies, e.g., Claeson, G., "Synthetic Peptides and Peptidomimetics as Substrates and Inhibitors of Thrombin and Other Proteases in the Blood Coagulation System", Blood Coag. Fibrinol. 5, 41 1-436 (1994).
• Several classes of anticoagulants currently used in the clinic are direct or indirect inhibitors of thrombin (heparin, low- molecular-weight heparin, coumarins, etc.).
• a prothrombinase complex comprising the protein (factor Xa) converts a proenzyme prothrombin into the active thrombin.
• Factor Xa belongs to a class of serine proteases and is formed from the protein (factor) X due to activation thereof. Unlike thrombin, which acts on a variety of protein substrates and specific receptors, factor Xa evidently acts on a single substrate, namely prothrombin. Since one molecule of factor Xa is able to generate up to 138 molecules of thrombin, direct inhibitors of protein Xa, as inhibitors of formation of thrombin may be used as efficient agents in the anticoagulant strategy. Therefore, it is obvious that agents, which selectively inhibit protein Xa, may be useful as in vitro diagnostic agents, or as therapeutic agents against diseases associated with coagulation.
• Polypeptides derived from hematophagous organisms may be efficient and inhibitors of factor Xa.
• United States Patent 4, 588, 587 describes the anticoagulant activity of the saliva of the Mexican leech, Haementeria officinalis.
• the active agent of this saliva is the polypeptide factor Xa inhibitor, antistasin (ATS), Another potent and highly specific inhibitor of Factor Xa, called tick anticoagulant peptide (TAP), has been isolated from the whole body extract of the soft tick Ornithidoros moubata.
• Factor Xa inhibitory compounds which are not polypeptides, have also been prepared, according to: Tidwell, R. R. et al.," Strategies for Anticoagulation With Synthetic Protease Inhibitors. Xa Inhibitors Versus Thrombin Inhibitors", Thromb. Res., 19, 339-349 (1980) ; Turner, A. D. et al.,”p-Amidino Esters as Irreversible Inhibitors of Factor IXa and Xa and Thrombin", Biochemistry, 25, 4929-4935 (1986); Hitomi, Y.
• Factor Xa inhibitors which are small molecule organic compounds, such as nitrogen containing heterocyclic compounds which have amidino substituent groups, wherein two functional groups of the compounds can bind to Factor Xa at two of its active sites.
• WO 99/10316 describes compounds having a 4-phenyl-N-alkylamidino-piperidin connected to a 3- amidinophenyl group via a carboxamidealkyleneamino bridge
• EP 798295 describes compounds having a 4-phenoxy-N-alkylamidino-piperidine group connected to an amidinonaphthyl group via
• Ri is selected from H, -CI, -F, -Br, -OH, -Me, -OMe;
• R 2 is selected from CH and N;
• R 3 and R4 are each independently selected from H, -CI, -F, -Br, -OH, -Me, -OMe;
• R 5 is selected from H or Q-Q alkyl which optionally contains hydroxyl, carboxyl, or ester groups;
• R ⁇ 5 is selected from:
• X ls X 2 , X 3 and t are each independently selected from H or C C 6 alkyl which optionally contains hydroxyl, carboxyl, or ester groups;
• R 8 , R-9 and R 10 are each independently selected from H, -CI, -F, -Br, -OH, -Me, -OMe;
• R 7 is selected from H, -CI, -F, -Br, -OH, -Me, -OMe or:
• Z 1; Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , Z 8 , Z 9 and Zio are each independently H or d-C 6 alkyl which optionally contains hydroxyl, carboxyl, or ester group;
• Another aspect of the invention is represented by a pharmaceutical composition for preventing or treating a condition in a mammal characterized by undesired thrombosis comprising a therapeutically effective amount of a compound of the invention.
• the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, a solvent, or diluent, which may the agents reported in Remington's "The Science and Practice of Pharmacy”, Kirk's Othmer's “Encyclopedia of Chemical Technology", and the monography "The Pharmacological Basis of Therapeutics, 3 rd Edition).
• One more aspect of the invention is represented by a method for preventing or treating a condition in a mammal characterized by undesired thrombosis comprising administering atherapeutically effective amount of a compound of formula (I).
• Conditions in a mammal characterized by undesired thrombosis are well known, in particular: acute coronary syndrome, myocardial infarction, unstable angina, refractory angina, thromboses associated with post-thrombolytic therapy or post-coronary angioplasty, a thrombotically mediated cerebrovascular syndrome, embolic stroke, thrombotic stroke, transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus, coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, thromboangiitis obliterans, thrombotic disease associated with heparin-induced thrombocytopenia, thrombo
• Yet one more aspect of the invention is represented by a method for inhibiting the coagulation of a biological sample comprising the step of administering a compound of formula (I).
• Amines (II) can be obtained by reduction of corresponding nitrocompounds (V) under appropriate conditions.
• a reduction agent for example, SnCl 2 or catalytic hydration on Ni-
• Raney can be used.
• Nitrocompounds (V) can be synthesized from corresponding amines (VI) and nitro- substituted carboxylic acids (VII)
• carboxylic acids (VII) are transformed into corresponding chloroanhydrides (VIII) by treatment with proper reagent such as SOCl 2 or POCl 3 .
• proper reagent such as SOCl 2 or POCl 3 .
• Chloroanhydrides (VIII) are then smoothly reacts with amines (VI) to yield nitrocompounds (V).
• An alternative method of synthesis of the amide bond may be a reaction of carboxylic acid (VII) with amine (VI) in the present of appropriate coupling agents, such as DCC, EDCI, and CDI.
• Urea derivatives of carboxylic acids can be synthesized by treatment of corresponding methyl or ethyl aminoesters (XI) with CDI followed by amine X 2 X 3 NH. Ethyl or methyl esters are then hydrolyzed in an alkaline medium to give desired compounds:
• aminoacid (Illb) can be transformed into corresponding urea
• a protective group for example, trifluoroacetyl group can be used.
• Protected amine (la) can be synthesized from the corresponding protected aminoacid (Hid) by coupling with - amine (II) in the presence of appropriate coupling agent.
• the protective group then sho ld be removed by appropriate agents; in the case of trifluoroacetyl group, NaOH can be used for this purpose.
• the amine (lb) can be also transformed into amidine (Id) by reaction with nitrile NC- CZ 2 Z 3 Z 4 in the presence of dry gaseous HCl.
• the aminine (Id) can be further alkylated or arylated to amidine (Ie) by appropriate alkyl- or aryl halogenide XI -Hal or other appropriate leaving group contained reagent.
• the reaction mixture is evaporated; the residue is dissolved in 30 ml of chloroform, rinsed with a saturated aqueous solution of NaHC0 3 ; the chloroform extract is evaporated; the residue is applied on a 40x150 mm column filled with 30 to 50 ⁇ of silica gel.
• the product is eluted with chloroform. Detection is carried out with the aid of an UV-unit at a wavelength of 280 nm. The UV absorbing fractions are collected; the purity of the product is controlled with a thin-layer chromatography technique in chloroform.
• the R f of the product is 0.4; the R f of the starting 2-amino-5-chloropyridine is 0.7.
• the 2-Nitrobenzoic acid in chloroform remains at the start (R f ⁇ 0.1).
• the yield of N-(5-chloropyridine-2-yl)-2-nitrobenzamide is 2.4 g.
• the aqueous fraction is extracted with chloroform; the extracts are joined together and evaporated.
• the residue is applied on a 30x150 mm column filled with 40 to 60 ⁇ ⁇ of silica gel.
• the product is eluted with chloroform. Detection is carried out with the aid of an UV-unit at a wavelength of 280 nm; the purity of the product is controlled with a thin-layer chromatography technique in chloroform.
• the R f of the product is 0.6; the yield of 2-amino-N-(5-chloropyridine-2-yl) benzamide is 650 mg.
• the chloroform extracts are joined together and evaporated.
• the residue is dissolved in water and applied on a 20x250 mm column filled with reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is rinsed with 100 ml of water; then, the elution with a gradient of ethyl alcohol of from 0 to 50% is carried out against a background of 1% acetic acid.
• the yield of the product is about per 30% of ethyl alcohol.
• N-(5-chloropyridine-2-yl)-2-[(4-ethaneimidoyl- methylaminophenylcarbonyl)amino]benzamide is 200 mg.
• aminosalicylic acid 1 g is dissolved in 20 ml of aqueous dioxane. The solution is added with 900 mg of di-tertbutyldicarbonate and 3 ml of 10% aqueous solution of NaOH. In 24 hours, the reaction mixture is evaporated, the 4-tertbutoxycarbonylaminosalicylic acid is purified by recrustallixation from ethanol. The yield is 1.1 g.
• a mixture of 0.5 g of 4-tertbutoxycarbonylaminosalicylic acid, 0.6 g of N-(3- chloropropyl)-acetamide, and 0.5 g of K 2 C0 3 is heated to 110°C; in an hour, the reaction mixture is cooled, diluted with water, and extracted with chloroform; the extract is evaporated and is applied onto a 30x150 mm column filled with 40-to 60 ⁇ of silica gel; the product is eluted with a 9:1 chloroform/ethanol mixture.
• R f 0.5 (9: 1 chloroform/ethanol).
• the yield of 2-[3-(acetylamino)propoxy]-4-[(tertbutoxycarbonyl)amino]benzoic acid is 400 mg.
• the chloroform-treated extracts are evaporated.
• the residue is dissolved in water and titrated with a 1% aqueous NaOH till pH 9.
• the solution is allowed to stand at 25°C for 3 hours; then it is neutralized, evaporated, the residue is applied onto a 20x250 column filled with a reversed phase C2 (RP2).
• RP2 reversed phase C2
• the column is washed with 100 ml of water; then the elution is carried out with a gradient of ethyl alcohol from 0 to 50% against the background of a 1% aqueous acetic acid.
• the product yields per approximately 30% or ethyl alcohol.
• the purity control is carried out with a TLC technique in a 8:2 isopropanol/aqueous ammonia system.
• the chloroform-treated extracts are joined together and evaporated.
• the solution is allowed to stand at 25°C for 3 hours, neutralized, evaporated, and the residue is applied onto a 2-x250 column filled with a reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is washed with 100 ml of water and then the elution is performed with an ethyl alcohol gradient of from 0 to 50% against the background of 1% aqueous acetic acid.
• the product yield is approximately per 30% of ethyl alcohol.
• the purity is controlled with a TLC technique in an 8:2 isopropanol/aqueous ammonia system.
• R f 0.5.
• N-(5-chloropyridine-2-yl)-2-[4-ethaneimidoylmethylamino)-2-(3- aminopropoxy)phenylcarbonyl-amino]-5-methyl-benzamide is 30 mg.
• the yield of N-(5-chloropyridine-2-yl)-2-[4-methylamino-2-(3- (acetylamino)propoxy)phenylcarbonylamino]-5-chlorobenzamide is 65 mg.
• the solution is allowed to stand at 25 °C for 3 hour, neutralized, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase C2 (RP2).
• RP2 reversed phase C2
• the column is washed with 100 ml of water; then elution is performed with a ethyl alcohol gradient of from 0 to 50% against the background of 1% aqueous acetic acid.
• the product yield is about per 30% of ethyl alcohol.
• N-(5-chloropyridine-2-yl)-2-[4- (ethaneimidoylmethylamino)-2-(3-aminopropoxy)phenylcarbonylamino]-5-chlorobenzamide is 30 mg.
• the yield of N-(5-chloropyridine-2-yl)-2-[4-(methylamino-2-(3- aminopropoxy)phenylcarbonylamino]-5-methoxybenzamide is 65 mg.
• the solution is allowed to stand at 25°C for 3 hours; then the solution is neutralized, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is washed with 100 ml of water, and elution is carried out with a gradient of ethyl alcohol from 0 to 50% against the background of 1% aqueous acetic acid.
• the yield of the product is about per 30% ethyl alcohol.
• N-(5-chloropyridine-2-yl)2-[4(ethaneimidoylmethylamino)-2- (3-aminopropoxy)phenylcarbonyl-amino]-5-methoxybenzamide is 30 mg.
• the yield of 2- [3 -(trifluoroacetamino)propoxy] -4- [(tert-butoxycarbonyl)methylamino] -6-fluorobenzoic acid is 390 mg.
• the yield of N-(5-chloropyridine-2-yl)-2-[4-methylamino-2-(3- aminopropoxy)-6-fluorophenylcarbonylamino]-5-methylbenzamide is 160 mg.
• the chloroform-treated extracts are joined together and evaporated.
• the solution is allowed to stand at 25°C for 3 hours, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase of C2 (FP2).
• the column is washed with 100 ml of water; then the elution is carried out with an ethyl alcohol gradient of from 0 to 50% against the background of 1% aqueous acetic acid.
• the product yield is about per 30% of ethyl alcohol.
• the product purity is controlled with a TLC technique in a 3:5:2 acetonitrile/dioxane/aqueous ammonia system.
• the yield of N-(5-chloropyridine-2-yl)-2-[4-methylamino-2-(3-aminopropoxy)-6- fluorophenylcarbonylamino]-5-chlorobenzamide is 160 mg.
• the chloroform-treated extracts are joined together and evaporated.
• the solution is allowed to stand at 25°C for 3 hours, neutralized, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is washed with 100 ml of water; then the elution is carried out with an ethyl alcohol gradient of from 0 to 50% against 1% aqueous acetic acid.
• the product yield is per about 30% of ethyl alcohol.
• the priduct purity is controlled with a TLC technique in a 3:5:2 acetonitrile/dioxane/aqueous ammonia system.
• R f 0.5.
• the yield of N-(5-chloropyridine-2-yl)-2-[2-(3-aminopropoxy)-6- fluoro- 4(ethaneamidoylmethylamino)phenylcarbonylamino]-5-chlorobenzamide is 60 mg.
• the yield of N-(5-chloropyridine-2-yl)-2-[2-fluoro-4- methylaminophenylcarbonylamino]-5-methylbenzamide is 380 mg.
• the solution is allowed to stand at 25°C for 3 hours, neutralized, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is washed with 100 ml of water; then the elution is carried out with an ethyl alcohol gradient of from 0 to 50% against the background of 1% aqueous acetic acid.
• the product yield is about per 30% of ethyl alcohol.
• N-(5-chloropyridine-2-yl)-2-[2-fluoro-4- ethaneimidoyl(methyl)amino)phenylcarbonylamino]5-methylbenzamide is 160 mg.
• the chloroform-treated extracts are joined together and evaporated.
• the solution is allowed to stand at 25°C for 3 hours, neutralized, evaporated, and the residue is applied onto a 2-x250 column filled with a reversed phase of C2(RP2).
• the column is washed with 100 ml of water; then the elution is carried out with a gradient of ethyl alcohol of from 0 to 50% against the background of aqueous acetic acid.
• the product yield is about per 30% of ethyl alcohol.
• N-(5-chloropyridine-2-yl)-2-[2-fluoro-4- (ethaneimidoyl(methyl)amino)phenylcarbonylamino]-5-chlorobenzamide is 140 mg.
• methylbenzamide is 80 mg.
• the solution is allowed to stand at 25°C for 3 hours, neutralized, evaporated, and the residue is applied onto a 20x250 column filled with a reversed phase of C2 (RP2).
• RP2 reversed phase of C2
• the column is washed with 100 ml of water and the elution is carried out with a gradient opf ethyl alcohol of from 0 to 50% against the background of 1% aqueous acetic acid.
• the product yield is about per 30% of ethyl alcohol.
• N-(5-chloropyridine-2-yl)-2-[4-(ethaneimidoylmethylamino)-2- (3-(acetylamino)propoxy)phenylcarbonylamino]-5-methylbenzamide is 20 mg.
• N-(5-chloropyridine-2-yl)-2-[(4- carbamoyl(methyl)aminophenylcarbonyl)amino]-5-methylbanzamide 60 mg are dissolved in 2 ml of THF and the solution is added with 200 ⁇ of dimethylsulfate. The reaction mixture is stirred for 24 hours, evaporated, and the residue is subjected to chromatographic separation on silica gel .
• a mixture of 0.5 g of 4-[tertbutoxycarbonyl(methyl)amino]salicylic acid, 0.6 g of N- (3-chloropropyl)-trifluoroacetylamide, and 0.5 g of K 2 C0 3 are heated to 1 10°C.
• the reaction mixture is cooled, dilutedwith water, and extracted with chloroform; the extract is evaporated and applied onto a 30x150 column filled with 40 to 60 ⁇ of silica gel; the product is eluted with a 9:1 chloroform/ethanol mixture.
• R f 0.5 (9:1 chloroform/ethanol).
• the yield of 2-[3-(trifluoroacetylamino)-propoxy]-4-[(tert- butoxycarbonyl)(methyl)amino]benzoic acid is 350 mg.
• N-(5-chloropyridine-2-yl)-2- ⁇ [4-methyl(methylcarbamoyl)- amino] -2- [3 -aminopropoxy]phenylcarbonyl ⁇ amino] -5 -methylbenzamide is 160 mg.
• N-(5-chloropyridine-2-yl)-2- ⁇ [4-methyl(methylcarbamoyl)- amino]-2-[3-aminopropoxy]phenylcarbonyl ⁇ amino]-5-chlorobenzamide is 160 mg.
• the protein and the active agent in the solution were under thermostatic control for 10 min; then the substrate was added and the reaction was initiated.
• the reaction rate was measured with a Specord M80 instrument from a change in the light absorption at a wavelength of 405 nm.
• the IC50 values were determined from the experimentally obtained dependence of the initial rates on the concentration of the inhibitor (active agent).
• the Kj values were calculated from the concentrations of the protein, substrate, and inhibitors; the ICs 0 values, by the method described in (1) Jordan, S.P.,; Waxman, L.; Smith, D.E.; Vlasik, G.P. Biochemistry 1990, 29, 1 1095 and (2) Morrison, J.F. Biochim. Biophys. Acta 1969, 185, 269. The results are shown in Table 1.

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